Association of smoking cigarettes, age, and sex with serum concentrations of olanzapine in patients with schizophrenia

Introduction Olanzapine is an atypical antipsychotic drug which is effective in the treatment of schizophrenia. Cigarette smoking, age, and sex could be related to the pharmacokinetics and serum concentrations of olanzapine in patients with schizophrenia. The aim of the study was to examine whether there was a significant difference in the serum olanzapine concentrations with regard to the mentioned factors. Materials and methods A total of 58 outpatients with schizophrenia (37 smokers, 42 men, 35 older than 40 years) participated in the study. Blood was sampled in serum tubes just before taking the next dose of olanzapine. Olanzapine was extracted by liquid-liquid extraction and was measured by an in-house high-performance liquid chromatography method on Shimadzu Prominence HPLC System with diode array detector SPD-M20A (Shimadzu, Kyoto, Japan). The results were expressed as the ratio of concentration to the daily dose of olanzapine (C/D). Non-parametric statistical tests were used to analyse differences between variables. Results The median C/D of olanzapine (interquartile range) in smokers was 6.0 (3.4-10.2) nmol/L/mg and in non-smokers 10.1 (5.9-17.6) nmol/L/mg; P = 0.007. The median C/D of olanzapine in patients younger than 40 years was 5.6 (4.5-10.2) nmol/L/mg and in patients older than 40 years 8.4 (5.6-13.0) nmol/L/mg; P = 0.105. The median C/D of olanzapine in male patients was 6.6 (4.6-10.4) nmol/L/mg and in female patients 9.0 (5.9-15.3) nmol/L/mg; P = 0.064. Conclusions The serum olanzapine concentration was significantly lower in smoking than in non-smoking patients with schizophrenia. No significant difference was demonstrated with regard to age and sex.


Introduction
Olanzapine is an atypical antipsychotic drug used in the treatment of patients with schizophrenia.It is effective in patients with a wide range of psy chopathology, including negative symptoms, has a lower frequency of extrapyramidal symptoms, and minimally affects prolactin concentrations (1).Olanzapine achieves its pharmacological effect through a combination of dopamine and seroto nin type 2 (5HT 2 ) receptor antagonism (2).How ever, weight gain and metabolic syndrome with the development of cardiovascular diseases are side effects responsible for shortened life expec tancy and poor adherence in patients with schizo phrenia (3).Although in clinical practice olanzap ine (and clozapine) have been associated with the highest risk of metabolic syndrome, their effec tiveness is still higher than of other atypical antip sychotics (3).For these reasons, olanzapine be longs to the group of antipsychotics (level 1) for which therapeutic drug monitoring (TDM) is strongly recommended (4,5). 2 Beside metabolic side effects, an additional prob lem in optimizing olanzapine dose is smoking giv en that 70 to 80% of patients with schizophrenia are smokers, with 36% of them smoking 20 ciga rettes or more per day (6,7).In clinical practice, dose adjustments may be necessary because smokers may be at risk of subtherapeutic psychop harmacological treatment (8).Olanzapine is exten sively metabolized by the cytochrome P4501A2 (CYP1A2) enzyme to the inactive product 4'des methyl olanzapine (9).Smoking can significantly affect the blood concentration of drugs that are CYP1A2 substrates.Polycyclic aromatic hydrocar bons (not nicotine) from cigarette smoke induce the CYP1A2 enzyme through activation of the aryl hydrocarbon receptor (AhR) and increase its activ ity (10).Consequently, this leads to a decrease in the olanzapine blood concentration and ineffec tive therapy (5,11).It is assumed that the olanzap ine serum concentration also may vary between individuals depending on sex and age.Women are known to have lower CYP1A2 enzyme activity and therefore lower olanzapine clearance than men (12).Some studies have shown that effect of age on serum olanzapine concentration becomes more pronounced in older age (12).Therapeutic drug monitoring is a valuable tool for personaliz ing the olanzapine dose in patients with schizo phrenia.The correct interpretation of the obtained results and optimization of pharmacotherapy us ing TDM reduces treatment cost and increases pa tient safety (2).
Because of the known large interindividual varia bility in olanzapine pharmacokinetics, we hypoth esized that age, sex, and lifestyle habits such as cigarette smoking might be associated with serum olanzapine concentrations in patients with schizo phrenia.The aim of our study was to examine whether there was a difference in the measured concentrations of olanzapine with regard to the smoking status, sex, and age of patients with schizophrenia.

Subjects
Fiftyeight outpatients who met schizophrenia di agnosing criteria listed in Diagnostic and statistical manual of mental disorders, 4th edition, partici pated in this study (13) pot formulation of olanzapine.The patients' blood was sampled in Vacuette tubes 4 mL CAT Serum Clot Activator (Greiner BioOne, Kremsmünster, Austria).The blood was centrifuged for 5 min at 3500 rpm at room temperature in a centrifuge Rotofix 32 (Hettich Zentrifugen, Tuttlingen, Ger many).Serum samples were kept in a refrigerator at + 4°C if the analysis was planned within 24 h.Se rum samples that were not analysed within 24 h were stored at 20°C and were analysed within a week.

Methods
Olanzapine (parent compound) was measured in serum samples by using an inhouse method on highperformance liquid chromatography (HPLC) with a diode array detector (DAD).The type of ana lyser was Shimadzu Prominence HPLC System with SPDM20A DAD (Shimadzu, Kyoto, Japan).The method was validated according to the Guideline on the validation of bioanalytical methods of the European medicines agency (14).Betweenday precision, expressed as coefficient of variation (CV), was 8.3% for level 1 (67.3 nmol/L) and 8.4% for level 2 (203.0 nmol/L), which fulfilled the vali dation criterion of < 10%.To assess the accuracy of the method, the recovery test was used which gave results of 99% for level 1 (67.3 nmol/L) and 101% for level 2 (203.0 nmol/L).The criterion for accuracy was ± 15% (85115%).The linearity of the method was in the concentration range from 4.8 to 960.0 nmol/L with the correlation coefficient r = 1.00 (criterion r > 0.99).The lower limit of quantifi cation (LLOQ) was 7.8 nmol/L and the limit of de tection (LOD) was 2.

Statistical analysis
The distribution of the collected data was evaluat ed with the D'AgostinoPearson test.The data did not follow a normal distribution (P < 0.001) and were presented as median and interquartile range.Nonparametric statistical tests were used to ana lyse differences between variables.The Mann Whitney test was used to examine differences be tween groups of patients divided by smoking sta tus, sex, and age.The KruskalWallis test was used to examine differences between groups of pa tients divided by the number of cigarettes smoked per day.The relationship between age and the smoking status of patients was examined using Spearman's rank correlation coefficient.The pro portion of nonsmokers and smokers with olan zapine concentrations below the therapeutic range was compared using the chisquare test.A value of P < 0.05 was taken as confirmation of a statistically significant difference.For statistical data analysis, MedCalc v20.110 (MedCalc Software Ltd, Ostend, Belgium) was used.The values of olanzapine serum concentrations (nmol/L) were divided by daily dose of drug (mg) to obtain con centration ratio to the daily dose of olanzapine (C/D) in nmol/L/mg, hereinafter referred to as doseadjusted concentration.The route influence of drug administration was avoided by using dose adjusted concentrations.Intramuscular doses of 300 and 405 mg/4 weeks, administered once a month, were converted to daily doses of 10 and 13.5 mg/day.Such uniform values were used in statistical tests.

Results
Median trough olanzapine concentrations in both smokers and nonsmokers were within therapeu tic range for olanzapine, but median trough olan zapine concentrations in smokers were much clos er to the lower limit of the therapeutic range (Ta ble 2).Approximately onethird of all patients had the trough olanzapine concentration below the lower limit of the therapeutic range (< 64 nmol/L), and more than twothirds of them were smokers.Using a chisquare test, it was found that the rela tion between smoking status and olanzapine se rum concentrations below therapeutic range was not significant, P = 0.278.However, median trough and doseadjusted olanzapine concentrations in smokers were approximately twice as low as in nonsmokers (Figure 1).There was a statistically significant difference between doseadjusted olanzapine concentrations in smokers and non smokers.Furthermore, there was no statistically significant difference between smokers depend ing on the number of cigarettes smoked per day.
On the right box and whisker graph in Figure 1, it is visible that C/D ratios of the first three groups of smokers decrease with an increase in the number of cigarettes smoked, but this decrease is not sig   nificant.Furthermore, patients smoking 2130 cig arettes per day and patients smoking more than 30 cigarettes per day had approximately similar median C/D of olanzapine (Table 2, Figure 1).
The influence of age and sex on serum olanzapine concentrations was also examined.The age limit of 40 years was taken as a criterion for dividing pa tients into two groups by age (Table 3).No differ ence was found between age groups, i.e. between patients younger and older than 40 years regard less of smoking status (Figure 2).Also, no correla tion was found between patients' age and smok      ing status (Spearman's coefficient correlation, r s = 0.17; P = 0.204).The difference between dosead justed olanzapine concentrations in male and fe male patients was not statistically significant (Fig ure 3, Table 4).

Discussion
The results of our study indicate that C/D of olan zapine in smokers is significantly lower than in nonsmokers.A higher C/D value indicates a slow metabolism of the drug and its removal, while lower C/D values indicate a rapid removal of the drug from circulation.It is known that smokers have a higher clearance and lower concentrations of olanzapine than nonsmokers (1).A metaanaly sis by Tsuda et al. showed that olanzapine doses should be reduced by 30% in nonsmokers com pared to smokers to obtain equivalent olanzapine concentrations (15).A population pharmacokinetic analysis showed that olanzapine clearance may be 37 to 48% higher in smokers (16).Furthermore, He res et al. have stated that olanzapine concentra tions are higher in nonsmokers than in smokers, regardless of olanzapine administration route (17).
Our results also agree with the results of the above mentioned studies.Further examination showed that the number of cigarettes smoked per day did not correlate with reduction in C/D of olanzapine.The metaanalysis by Djordjevic et al. found that smoking at least five cigarettes per day was associ ated with reduced efficacy of olanzapine (11)  7 tion of CYP1A2 is achieved after smoking 10 ciga rettes per day and that smoking 10 or 30 cigarettes per day does not cause a difference in serum con centrations of olanzapine (10).Additionally, Carrillo et al. found that nonsmokers and those who smoke four or less cigarettes per day had similar CYP1A2 activity (9).The consensus guidelines pub lished by Hiemke et al. state that the effects of smoking should be taken into account when smoking more than 10 cigarettes per day (4).Se rum olanzapine concentrations below therapeutic range are not related to the patients' smoking sta tus.Our finding is consistent with the fact that there are no guidelines in the literature for adjust ing the olanzapine dose according to smoking sta tus.
The median C/D of olanzapine in patients older than 40 years is not significantly higher than in pa tients younger than 40 years.It is assumed that concentration of olanzapine will be higher the old er the patient is (12).In this study, an arbitrary cut off value of 40 years was taken, so older patients were considered to be those over 40 years old.The oldest patient was 64 years old, so the group of el derly patients was heterogeneous.A study by Castberg et al. which included more than 3000 pa tients (the oldest patient was 86 years old) showed that every five years after the age of 40, dosead justed concentration of olanzapine increased by 1.7% in men and 1.3% in women (12).Another study has found an increase of about 27% in dose adjusted olanzapine concentrations in patients over 60 years of age compared with younger sub jects.It should be noted that studies examining the effect of age on olanzapine concentrations are limited due to the small number of very old pa tients, over 85 years of age (12).This was also the case in our study.Some authors state that olan zapine prescription rates decrease slightly in elder ly patients, and if olanzapine is prescribed, a lower starting dose (5 mg) is recommended (19).In el derly people, the activity of the CYP1A2 enzyme remains preserved but organ perfusion (liver and kidney) and circulation weaken so that metabo lism and elimination of olanzapine can be slowed down (4).The most likely mechanism underlying the higher doseadjusted concentrations in the el derly is a lowered hepatic clearance of the drugs caused by a decreased intrinsic hepatic metabolic function, lower liver volume, and/or reduced he patic blood flow due to, for example, impaired car diac output (12).The drug metabolism may also be altered due to kidney and liver diseases.
In our study, no statistically significant difference was obtained in doseadjusted olanzapine con centrations according to sex.In an extensive study based on the results from the routine TDM data base (16,171 samples for olanzapine), it was shown that doseadjusted olanzapine concentrations in women were 26.1% higher than in men (12).The reason for the higher olanzapine concentration dose ratio in women may be that CYP1A2 also me tabolizes some female sex hormones (20).There fore, there is competition between CYP1A2 sub strate drugs and hormones.This observation is most pronounced in late pregnancy (20).In our study, there were no data related to the pregnan cy of patients.Another fact is that olanzapine ab sorption is higher in women than in men due to slower gastric emptying and a longer gastrointes tinal transit time (21).In addition, the slower elimi nation and higher volume of olanzapine distribu tion in women lead to a longer halflife of olanzap ine and slower clearance (21).Other possible mechanisms include sex differences in blood flow and liver size, as well as differences in the expres sion of metabolizing enzymes and transporters

Figure 1 .
Figure 1.On the left: doseadjusted concentrations (C/D) of olanzapine in nonsmokers and smokers.On the right: doseadjusted concentrations (C/D) of olanzapine in smokers with regard to the number of cigarettes smoked per day.The boxes represent the in terquartile range, the middle horizontal line the median, and the vertical line minmax values.

Figure 2 .
Figure 2. Doseadjusted concentrations (C/D) of olanzapine in patients with schizophrenia considering the age limit of 40 years.The boxes represent the interquartile range, the middle horizontal line the median, and the vertical line minmax values.

Figure 3 .
Figure 3. Doseadjusted concentrations (C/D) of olanzapine in male and female patients with schizophrenia.The boxes repre sent the interquartile range, the middle horizontal line the me dian, and the vertical line minmax values.

Table 1 .
Groups of patients whose blood was analysed in the study with regard to sex, age, route of drug administration, and smok ing status Variables are presented as median (interquartile range).Differences between doseadjusted concentrations of olanzapine in nonsmokers and smokers were determined by the MannWhitney U test, while differences between smokers with regard to the number of cigarettes smoked were determined by the KruskalWallis test.C trough concentrations of olanzapine.C/D doseadjusted concentrations of olanzapine.P < 0.05 was considered statistically significant.

Table 2 .
Results obtained for nonsmokers and smokers

Table 3 .
Results obtained considering the age limit of 40 years